The action of CD4+CD25 + T regulatory (Treg) cells represents a major mechanism to inhibit self-reactivity by suppressing autoreactive mature peripheral T cells that have escaped thymic negative selection. In spite of the ready ability to demonstrate this immunological phenomenon, comparatively little is known concerning the factors controlling Treg cell development, specificity, homeostasis, and function. Recently, our laboratory established one important principle concerning Treg cells, namely that IL-2 is essential for their development. Importantly, we showed that the adoptive transfer of CD4+CD25+Treg cells prevented the onset of the rapid and lethal autoimmune disease in IL-2Rbeta -/- mice that was accompanied by substantial expansion, followed by long-term stable engraftment, using syngeneic or fully allogeneic donor Treg cells. This result makes plain that Treg cells have considerable growth potential in vivo. Therefore, one major objective of this project is to define the mechanism responsible for the proliferation and homoeostasis of Treg cells. The efficacy of allogeneic Tregs cells to prevent autoimmunity is of considerable practical value in developing strategies to utilize Treg cells in adoptive immunotherapy. Furthermore, as will be shown in the Preliminary Data, many Treg cells are specific for allogeneic MHC, as these cells have been selected to expand and survive in MHC-mismatched recipient mice. In principle, therefore, the TCR specificity is defined for these T reg cells, i.e. alloreactive to MHC class II. Thus, along with characterizing the immunosuppressive properties of allogeneic Tregs cells, the other major objective of this proposal is to utilize this and other model systems to investigate the specificity and diversity of the TCR as it relates to Treg cell homeostasis and suppressive activity. The specific aims are: 1) To further define the basis by which allogeneic CD4+CD25vTreg cells prevent autoimmunity in IL-2Rbeta-/- mice; 2) to characterize the growth and survival characteristics of CD4+CD25+Treg cells in vivo; and 3) to test the effect of limiting the diversity of the TCR repertoire on the development and function of Treg cells.